Chimeric bacteriophage lysin with activity against staphylococci bacteria

ABSTRACT

The present description relates to chimeric bacteriophage lysins useful for the identification and/or reduction of staphylococcal populations. For example, a chimeric bacteriophage lysin was engineered and shown to effectively kill all strains of staphylococci tested including antibiotic resistant methicillin-resistant S. Aureus and VISA.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 61/078,277 filed on Jul. 3, 2008, which is herein incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under grant number A111822 awarded by the National Institutes of Health (NIH). The U.S. government may retain certain rights to the invention.

TECHNICAL FIELD

The present disclosure relates to the identification and use of chimeric lytic enzymes to rapidly and specifically detect and kill Staphylococci bacteria, including certain antibiotic-resistant Staphylococcus aureus bacterial strains.

BACKGROUND

Staphylococcus aureus is an opportunistic pathogen inhabiting human skin and mucous membranes. S. aureus is the causative agent of variety of skin and soft tissue infections in humans and serious infections such as pneumonia, meningitis, endocarditis, and osteomyelitis. S. aureus exotoxins also cause disease syndromes such as bullous impetigo, scalded skin syndrome, and toxic shock syndrome. Additionally, staphylococci are also among the most common causes of food-borne illness in United States (Fischetti V A, Novick, R. P., Ferretti, J. J., Portnoy, D. A. and Rood, J. I., editor. 2006. Gram-positive pathogens. 2nd ed: ASM Press). S. aureus is also a major cause of community- and hospital-acquired (nosocomial) infections. Of the nearly 2 million cases of nosocomial infections in United States, approximately 230,000 cases are caused by S. aureus (NNIS. 2003. NNIS report, data summary from January 1992 through June 2003, issued August 2003. American Journal of Infection Control 31:481-498).

The global appearance of methicillin- and vancomycin-resistant clinical isolates of S. aureus has become a serious concern. Currently, 40-60% of nosocomial infections of S. aureus are resistant to oxacillin (Massey R C, Horsburgh M J, Lina G, Hook M, Recker M. 2006. The evolution and maintenance of virulence in Staphylococcus aureus: a role for host-to-host transmission? Nat Rev Microbial 4(12):953-8.) and greater than 60% of the isolates are resistant to methicillin (Gill S R, Fouts D E, Archer G L, Mongodin E F, Deboy R T, Ravel J, Paulsen I T, Kolonay J F, Brinkac L, Beanan M and others. 2005. Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. J Bacterial 187(7):2426-38). Treating infections caused by the drug-resistant S. aureus has become increasingly difficult and therefore is a major concern among healthcare professionals. To combat this challenge, development of new and effective antibiotics belonging to different classes are being aggressively pursued. A number of new antimicrobial agents such as linezolid, quinupristin-dalfopristin, daptomycin, tigecyline, new glycopeptides and ceftobiprole have been introduced or are under clinical development (Aksoy D Y, Unal S. 2008. New antimicrobial agents for the treatment of Gram-positive bacterial infections. Olin Microbiol Infect 14(5):411-20). However, clinical isolates of MRSA (methicillin-resistant Staphylococcus aureus) with resistance to these new classes of antibiotics have already been reported (Tsiodras S, Gold H S, Sakoulas G, Eliopoulos G M, Wennersten C, Venkataraman L, Moellering R C, Ferraro M J. 2001. Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet 358(9277):207-8; Mangili A, Bice I, Snydman D R, Hamer D H. 2005. Daptomycin-resistant, methicillin-resistant Staphylococcus aureus bacteremia. Clin Infect Dis 40(7):1058-60; Skiest D J. 2006. Treatment failure resulting from resistance of Staphylococcus aureus to daptomycin. J Olin Microbial 44(2):655-6). Consequently, there is an urgent need to develop novel therapeutic agents or antibiotic alternatives against MRSA.

Bacteriophage endolysins (lysins) are one such class of novel antimicrobial agents that are emerging as novel agents for the prophylactic and therapeutic treatment of bacterial infections. Lysins are cell wall hydrolases that are produced during the infection cycle of double-stranded DNA bacteriophages (or phages) enabling release of progeny virions. Typically, lysins have two distinct functional domains consisting of a catalytic domain for peptidoglycan hydrolysis and a binding domain for recognition of surface moieties on the bacterial cell walls. The catalytic domains are relatively conserved among lysins. The activities of lysins can be classified into two groups based on bond specificity within the peptidoglycan: glycosidases that hydrolyze linkages within the aminosugar moieties and amidases that hydrolyze amide bonds of cross-linking stem peptides. The binding domains however are not conserved among lysins. Hence the binding domain imparts species- and strain-specificity because the binding targets, often carbohydrates associated with the peptidoglycan, display species- or strain-specific distribution (Fischetti V A, Nelson D, Schuch R. 2006. Reinventing phage therapy: are the parts greater than the sum? Nat Biotechnol 24(141508-11), The modular architecture of lysins' is an important feature with respect to their development as antimicrobial agents. This enables creation of chimeras by swapping lysin domains and thereby altering binding specificity or enzymatic activity or both (Sheehan M M, Garcia J L, Lopez R, Garcia P. 1996. Analysis of the catalytic domain of the lysin of the lactococcal bacteriophage Tuc2009 by chimeric gene assembling. FEMS Microbiol Lett 140(1):23-8; Lopez R G E, Garcia P, Garcia J L. 1997. The pneumococcal cell wall degrading enzymes: a modular design to create new lysins? Microb Drug Res 3:199-211; Croux C, Ronda C, Lopez R, Garcia J L. 1993. Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal-clostridial cell wall lytic enzyme. Mol Microbiol 9(5):1019-25; Donovan D M, Dong S, Garrett W, Rousseau G M, Moineau S, Pritchard D G. 2006. Peptidoglycan hydrolase fusions maintain their parental specificities. Appl Environ Microbial 72(4):2988-96).

When applied exogenously, native or recombinant lysins were able to degrade the cell wall of susceptible bacteria and cause rapid cell lysis (Nelson D, Loomis L, Fischetti V A. 2001. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc Natl Aced Sci USA 98(7):4107-12). Lysins have been developed against a number of Gram-positive pathogens including Group A streptococci (Nelson D, Loomis L, Fischetti V A. 2001. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc Natl Aced Sci USA 98(7):4107-12), S. pneumoniae (Loeffler J M, Nelson D, Fischetti V A. 2001. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294(5549):2170-2), Bacillus anthracis (Schuch R, Nelson D, Fischetti V A. 2002, A bacteriolytic agent that detects and kills Bacillus anthracis. Nature 418(6900):884-9), enterococci (Yoong P, Schuch R, Nelson D, Fischetti V A. 2004. Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium. J Bacterial 186(14):4808-12), Group B streptococci (Cheng Q, Nelson D, Zhu S. Fischetti V A. 2005, Removal of group B streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme. Antimicrob Agents Chemother 49(1):111-7), and Staphylococcus aureus (Rashel M, Uchiyama J, Ujihara T, Uehara Y, Kuramoto S, Sugihara S, Yagyu K, Muraoka A, Sugai M, Hiramatsu K and others, 2007. Efficient elimination of multidrug-resistant Staphylococcus aureus by cloned lysin derived from bacteriophage phi MR11. J Infect Dis 196(8):1237-47). The activities of most of these lysins have been demonstrated in vitro and in in vivo models. Several unique characteristics of lysin make them attractive antibacterial candidates against Gram-positive pathogens. These include i) rapid antibacterial activity both in vitro and in vivo; ii) very narrow lytic spectrum (species- and strain-specific); iii) very strong binding affinity, typically in the nanomolar range; iv) very low chances of developing resistance since the binding epitopes are essential for viability; v) safe; and vi) relative ease of modification by genetic engineering (Fischetti V A, Nelson D, Schuch R. 2006. Reinventing phage therapy: are the parts greater than the sum? Nat Biotechnol 24(12):1508-11).

Although lysins have been developed against a number of Gram-positive pathogens, there remains a need for a S. aureus-specific lysin. Various labs have unsuccessfully attempted to obtain a staphylococcal lysin. The expression of more than twenty different staphylococcal lysins using a variety of techniques have been attempted without success. These include expression of lysin genes in E. coli using different expression vectors and conditions, expression in Bacillus, yeast and mammalian systems, expression in the presence of chaperones, expression of truncated versions etc. To our knowledge, there is only one report of the successful development of S. aureus-specific lysin called MV-L (Rachel M, Uchiyama J, Ujihara T, Uehara Y, Kuramoto S, Sugihara S, Yagyu K, Muraoka A, Sugai M, Hiramatsu K and others. 2007. Efficient elimination of multidrug-resistant Staphylococcus aureus by cloned lysin derived from bacteriophage phi MR11. J Infect Dis 196(8):1237-47). MV-L lysin is comprised of two catalytic domains (an endopeptidase and an amidase domain) linked to a single cell wall targeting (CWT) domain, a type of binding domain. Unless otherwise indicated, references herein to a “binding domain” herein include a CWT domain. The MV-L CWT domain, like the staphylolytic enzyme lysostaphin, displays homology to SH3b-like domains. The SH3b-like domains bind to the peptide cross-bridge (the penta Glycine) in the staphylococcal cell wall. There are reports of staphylococcal strains developing resistance at 10⁻⁶ frequencies to lysostaphin by altering their peptide cross-bridges. Therefore, we expect staphylococci to develop resistance at a higher frequency to lysins containing SH3b-like CWT domains including MV-L. There is a need for lytic enzymes capable of specific binding to Staphylococcal bacteria without undesirably high frequencies of lysostaphin resistance, such as S. aureus specific lysins without SI-13b-like CWT domains.

SUMMARY

This disclosure describes novel staphylococcal lysins, as well as methods of making and using the lysin. In one example, the genetic engineering of a novel chimeric lysin called ClyS (for chimeric lysin for staphylococci) is described. ClyS is specifically active against susceptible and drug-resistant staphylococci, and was constructed by fusing the catalytic domain of a Staphylococcus-specific phage lysin with a unique binding domain from another Staphylococcus-specific phage lysin that has no known homologs. ClyS is a soluble Staphylococcal-specific lysin without a SH3b-like CWT domain, but does contain a CWT domain that is believed to recognize a staphylococci-specific surface carbohydrate. Consequently, the frequency by which staphylococcal strains will develop resistance to ClyS may be reduced. Additionally, biochemical characterization of ClyS revealed that the pH and salt spectrum of ClyS is very different from conventional lysins thereby providing unique properties to this chimeric lysin.

Also included within the scope of the present invention are methods of using the binding domain for diagnostic purposes, the method comprising the steps of contacting a sample with a reporter molecule comprising a cell wall target domain comprising the amino acid sequence of SEQ ID NO:1 and a fluorescent reporting moiety bound thereto; and subsequently detecting the presence of the reporter molecule bound to a staphylococcus bacteria within the sample. In certain embodiments, the reporter molecule is a green fluorescent protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of phiNM3 lysin showing the putative CHAP (“cysteine- and histidine-dependent amidohydrolase/peptidase”) and CWT domains. The numbers represent the amino acid positions and the domain limits. The CWT domain of ClyS is indicated in the diagram.

FIG. 2A is a gel showing the purification of phiNM3 CWT. SDS-PAGE and coornassie blue stained gel of phiNM3 CWT purified by anion-exchange chromatography is depicted in the lane marked “CWT.” Protein molecular weight markers in kilodaltons (kDa) are shown in the lane marked “M.”

FIG. 2B shows the amino acid sequence of the phiNM3 CWT protein (SEQ ID NO:1).

FIG. 3 shows a series of micrographs showing PhiNM3 CWT binding specifically to staphylococci. Purified phiNM3 CWT was labeled with FITC and exposed to 1) S. aureus; 2) B. cereus; 3) S. epidermidis; 4) E. coli; 5) Group A Streptococcus and 6) mixed suspension of S. aureus and B. cereus cells. “P” indicates phase-contrast image and “F” indicates fluorescent image.

FIG. 4 is a schematic diagram illustrating chimeric lysin development. In particular, FIG. 4 provides schematic diagrams of various chimeric lysins showing their respective domains and the corresponding expression and solubility of the protein and activity against S. aureus Similar domains are depicted in the same shading and labeled. PlyB-cat indicates catalytic domain of Bacillus-specific lysin PlyB (and is marked with a “4” in the figure); Sa-aa indicates 16 amino acid residues specific for staphylococcal lysins (and is marked with a “5” in the figure); PlyB-CWT indicates CWT domain of PlyB (and is marked with a “6” in the figure); Twort-CWT indicates CWT domain of S. aureus phage Twort lysin (and is marked with a “8” in the figure); Lysostaphin CWt indicates CWT domain of lysostaphin (and is marked with a “10” in the figure); and Se autolyin amidase indicates an amidase domain of S. epidermidis autolysin (and is marked with a “12” in the figure).

FIG. 5A shows the ClyS protein sequence. The predicted protein sequence of the chimeric protein ClyS showing the Twort endopeptidase catalytic and the phiNM3 CWT domains.

FIG. 5B shows the amino acid sequence for the AD127 chimeric molecule, described with respect to FIG. 4.

FIG. 5C shows the amino acid sequence for the native (unmodified) Twort lysin (SEQ ID NO: 12).

FIG. 6 is a gel showing the purification of ClyS. ClyS was expressed in E. coli DH5α cells and purified by cation-exchange chromatography followed by hydroxyapatite chromatography. Purified sample (10 micrograms) was separated by SDS-PAGE and stained by Coomassie blue (right hand lane). Protein molecular weight markers in kilodaltons (kDa) are shown in the left hand lane.

FIG. 7 is a graph showing the activity of ClyS against S. aureus in vitro. S. aureus strain 8325-4 cells were resuspended in 20 mM phosphate buffer (pH 7.4), incubated with 50 U of ClyS and OD600 (filled triangles) monitored by a spectrophotometer. Control experiments (filled squares) were performed under the same conditions with buffer alone. Viability (filled diamonds) of cells, shown as colony-forming units/ml, was determined by serially diluting and plating the cells.

FIG. 8 is a series of micrographs showing that ClyS causes cell wall disruption and ultimately lysis of 8325-4 cells. FIGS. 8A-8C (A-C) are thin-section transmission electron micrographs (bars, 200 nm) of S. aureus 3 minutes after exposure to 50 U of ClyS. The arrows indicate cytoplasmic membrane extrusions through holes generated in the cell wall by ClyS. Ultimate lysis results in “cell-ghosts” (D) after the loss of cytoplasmic contents (bar, 500 nm).

FIGS. 9A and 9B are graphs showing the activity of ClyS in various pH and salt concentration conditions. FIG. 9A is a graph of the activity of ClyS (50 U) tested against S. aureus strain 8325-4 in buffers with pH values ranging from 4 and 10 in 15 minute assays. Optical density (filled squares) and viability (filled diamonds) was measured as described in legend of FIG. 6. Fold killing in the viability assay was calculated by dividing the number of viable bacteria after buffer treatment at a particular pH by the number after exposure to ClyS enzyme at the same pH. Final pH readings for each reaction are recorded on the x axis. FIG. 9B is a graph showing the activity of ClyS (50 U) tested against S. aureus strain 8325-4 in 20 mM phosphate buffer (pH 7.4) in the presence of different concentrations of NaCl, After 15 minutes samples were assayed for optical density and viability calculated as above.

FIG. 10 is a bar graph showing that ClyS exerts specific killing of staphylococci. Log-phase cultures of different bacteria were exposed to 50 U of ClyS for 15 minutes. Fold killing was calculated as described in FIG. 8 legend.

FIG. 11 depicts a graph of the CFU of MRSA from individual MRSA infected mice after being administered phosphate buffered saline pH 7.3 (control) or ClyS (630 μg).

FIG. 12 depicts Kaplan Meier Survival Curves showing the effect of ClyS on preventing death in mice injected with MRSA compared with phosphate buffer control.

FIG. 13 depicts an isobologram for a checkerboard broth microdilution study of the effect of vancomycin on VISA (vancomycin-resistant Staphylococcus aureus) or oxacillin on MRSA with increasing amounts of ClyS.

FIG. 14 depicts Kaplan Meier Survival Curves showing the effect of oxacillin alone or in combination with ClyS.

FIG. 15 depicts a photograph of a Coomassie-blue stained SOS-PAGE gel of a 5-day time-course at 21° C. of ClyS in the absence (top left gel) or presence of 5 mM OTT (top right gel), and pClyS in the absence (bottom left gel) or presence of 5 mM DTT (bottom right gel). About 20 micrograms of protein was loaded into each lane of the gel. The bottom right gel shows a much higher amount of intact pClyS in the presence of 5 mM DTT after 5 days compared to intact ClyS in the presence of 5 mM DTT after 5 days.

DETAILED DESCRIPTION Definitions

Unless otherwise indicated, the certain terms used herein and their applicability to the present disclosure are defined below.

The term “isolated” means at least partially purified from a starting material. The term “purified” means that the biological material has been measurably increased in concentration by any purification process, including by not limited to, column chromatography, HPLC, precipitation, electrophoresis, etc., thereby partially, substantially or completely removing impurities such as precursors or other chemicals involved in preparing the material. Hence, material that is homogenous or substantially homogenous (e.g., yields a single protein signal in a separation procedure such as electrophoresis or chromatography) is included within the meanings of isolated and purified. Skilled artisans will appreciate that the amount of purification necessary will depend upon the use of the material. For example, compositions intended for administration to humans ordinarily must be highly purified in accordance with regulatory standards.

The term “lytic enzyme genetically coded for by a bacteriophage” refers to a polypeptide having at least some lytic activity against the host bacteria.

Variants of “chimeric bacteriophage lysin” are included within the definition of chimeric bacteriophage lysins, and include a functionally active chimeric bacteriophage lysin with killing activity against Staphylococcus aureus having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or even at least 99.5% amino acid sequence identity with a sequence described herein. For example, the present invention includes chimerical bacteriophage lysins having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or even at least 99.5% amino acid sequence identity with the polypeptide sequence of SEQ ID NO:2.

“Percent (%) polypeptide sequence identity” with respect to the lytic enzyme polypeptide sequences identified here is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific lytic enzyme polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Methods for alignment for purposes of determining percent amino acid sequence identity are described below.

Staphylococcal Lysins

Chimeric bacteriophage lysins with killing activity against S. aureus are described herein. Lysins generally occur in a modular structure. FIG. 1 is a schematic diagram of phiNM3 lysin showing the putative CHAP domain 110 and the CWT domain 120. The numbers represent the amino acid positions and the domain limits. The CWT domain of ClyS is shown as shaded box 120. The N-terminal module consists of a catalytic domain believed to possess the ability to break down the bacterial cell wall of certain bacteria. Enzymatic activities often associated with the catalytic domain are amidases, endopeptidases, glucosamidases and muramidases. The C-terminal module consists of a binding domain that is believed to have an affinity for a carbohydrate epitope on the target bacteria cell wall. The binding domain is believed to determine the specificity of the lysin. The peptide cross-bridge within the staphylococcal peptidoglycan is believed to function as the receptor for the CWT domain of lysostaphin, a staphylolytic enzyme produced by Staphylococcus simulans. The CWT domain of lysostaphin has homology to the SH3b domain suggesting that such lysins might also utilize the peptide cross-bridge as its receptor.

In one embodiment, Staphylococcus-specific binding molecules comprising a CWT domain within staphylococcal lysins are provided that have no known domain homologs. In some embodiments, the binding molecules are lysins. In other embodiments, the binding molecules may be used as diagnostic tools, for example to identify the presence of Staphylococcus bacteria. Preferably, such a CWT domain is provided to recognize a different epitope such as a cell wall-associated carbohydrate instead of the peptide cross-bridge in the staphylococcal cell wall.

In a further embodiment, the ClyS lysine can be used to digest the cell wall of Staphylococcus aureus bacterial strains, which in turn would allow access to the genetic and cytoplasmic material, such as endogenous DNA and RNA, to further identify and sequence the Staphylococcus aureus bacterial strain. It will also release membrane-associated and wall-associated molecules for diagnostic purposes.

Most preferably, the binding molecule is a soluble binding domain of a bacterial lysin comprising a polypeptide including an amino acid sequence providing specific binding to S. aureus, such as SEQ ID NO:1 (phiNM3 CWT domain). For example, the lysin preferably includes the polypeptide sequence of S. aureus phage phiNM3 lysin (SEQ ID NO:1) (protein accession number YP_908849). The phiNM3 lysin CWT domain (SEQ ID NO:1) corresponding to amino acid residues 158-251 was cloned and expressed. The approximately 10-kDa protein of SEQ ID NO:1 was highly soluble and was purified by one-step anion-exchange chromatography to homogeneity. FIG. 2A is an anion exchange gel showing the protein of SEQ ID NO:1 in a second column next to a set of marker proteins in a first column. FIG. 2B shows the amino acid sequence of SEQ ID NO:1. To determine whether the peptide domain of SEQ ID NO:1 displayed Staphylococcus-specific binding, the purified protein was labeled with FITC and exposed to log-phase S. aureus, S. epidermidis and mixed population of S. aureus and Bacillus. Group A streptococci, E. coli and Bacillus cereus were used as controls. More preferably, The FITC-labeled phiNM3 CWT domain bound specifically to S. aureus (FIG. 3-1) and S. epidermidis (FIG. 3-3) cells when present in single or mixed populations (FIG. 3-6) while binding to streptococci (FIG. 3-5), Bacillus (FIG. 3-2) or E. coli (FIG. 3-4) was not observed. PhiNM3 lysin specifically bound to S. aureus (FIG. 3-1) and S. epidermidis (FIG. 3-2) cells when present in single or mixed populations (FIG. 3-3) while binding to streptococci (FIG. 3-4), Bacillus (FIG. 3-5) or E. coli (FIG. 3-6) was not observed.

In one embodiment, the binding molecule comprises a CWT binding domain, such as the amino acid sequence of SEQ ID NO:1, attached to a reporting portion that is detectable to identify the presence of the binding molecule bound to Staphylococcal bacteria. For example, the binding molecule may include the amino acid sequence of SEQ ID NO:1 bound to a fluorescent reporter group, a radioactive reporter group or a heterologous tag that is adapted to bind a fluorescent reporter. The phiNM3 (SEQ ID NO:1) CWT domain may be used as a diagnostic tool for the identification of staphylococcal bacteria. The high affinity binding site may be used in a wide range of assay techniques to detect S. aureus. Such assay methods include radioimmunoassays, gold sol radial immune assays, competitive-binding assays, Western Blot assays and ELISA assays. Such detection assays advantageously utilize a heterogeneous format wherein a binding reaction (SEQ ID NO:1) between a conjugated binding agent comprising (SEQ ID NO:1) and an analyte occurs followed by a wash step to remove unbound conjugated binding agent. For example, gold sol particles may be prepared with protein that comprises the binding region with the binding protein immobilized on the particle surfaces. As binding occurs between the protein and (staphylococcal) bacteria, the particles merge and form a colored product. Analogously, the binding protein may be complexed, preferably covalently with an enzyme such as beta galactosidase, peroxidase, or horseradish peroxidase. After wash, the remaining bound enzyme can be detected by adding a substrate such as a fluorogenic or chemilumigenic substrate. The binding protein may be complexed with any other reagent that can make a signal such as a rare earth fluor and detected by time resolved fluorescence, a radioactive material and detected by radioactivity measurement, green fluorescent protein (GFP) or another fluorescent tag, and detected by fluorescence.

For comparison, FIG. 5B provides the amino acid sequence of SEQ ID NO:3, the AD119 sample discussed with respect to FIG. 4. AD119 (SEQ ID NO:3) comprises the Twort endopeptidase domain joined to the Lysostaphin CWT domain. In contrast to the chimeric compound of SEQ ID NO:2 (AD127), which shares the Twort endopeptidase domain but has the phiNM3 CWT domain (SEQ ID NO:1) in place of the Lysostaphin CWT domain, the AD127 compound was insoluble and exhibited little or no killing activity against S. aureus.

The conjugation of the binding region with a detectable tag may be carried out by synthetic chemistry or a biological process. For example, a DNA sequence coding for the binding region of SEQ ID NO:1 or of the entire lysin of SEQ ID NO:2 can be linked to genetic information that encodes a detectable marker such as green fluorescent protein (GFP) or an enzyme such as alkaline phosphatase. This could be accomplished by separating the DNA for the binding domain by removing the N-terminal catalytic domain and replacing it in frame with indicator molecules such as green flourescent protein (GFP) and purifying the expressed fusion molecule for the identification of S. aureus. Since the binding domain has a similar binding affinity of an immunoglobulin G molecule, the marked binding domain will effectively identify Staphylococcus aureus with little false positive activity. One also could fuse the GFP molecule or an enzyme at the 5′ end of the whole lysin enzyme if necessary, by doing so the enzymatic domain will be at least partly inactivated, still allowing the binding domain to function to bind to its substrate in the bacillus cell wall. Optionally, the <isolated binding domain of SEQ ID NO:1 may be separated from the catalytic domain of SEQ ID NO:2 and may be expressed, purified and labeled using a number of fluorescent molecules such as fluorescein isothiocyanate, rhodamine isothiocyanate and others known by skilled artisans. The binding domain may be modified with biotin to allow formation of a biotin-avidin complex after the binding region adheres to the Staphylococcus aureus for identification.

In another embodiment, the lysin is a chimeric protein that comprises an endopeptidase domain of the S. aureus Twort lysin upstream of the phiNM3 CWT domain (SEQ ID NO:1). The chimeric polypeptide is preferably sufficiently soluble in phosphate buffered saline (PBS). Preferred levels of solubility in PBS for the chimeric lysins is at least about 1 mg/ml and more preferably at least about 3 mg/mL. in PBS. While native staphylococcal bacteriophage lysins are typically insoluble in PBS, the chimeric lysins comprising an endopeptidase domain of a first lysin (e.g., Twort S. aureus lysin) bound to the CWT domain of SEQ ID NO:1 are surprisingly soluble in PBS (e.g., at least about 1 mg/ml, and typically about 3 mg/ml or greater). One example of such a lysin is provided in SEQ ID NO:2 (AD127), shown in FIG. 5A and consisting of the Twort lysin endopeptidase domain attached to the phiNM3 CWT domain (SEQ ID NO:1). The isolated polypeptide of SEQ ID NO:2 (AD 127) was constructed by engineering S. epidermidis autolysin amidase and Twort lysin endopeptidase domains upstream of phiNM3 CWT domain, respectively. Chimera AD 126 had no expression or activity but AD 127 was soluble and had very high activity but low expression. To overcome low expression of AD 127 construct, the entire chimera gene was cloned into expression vector pJML6 to generate pAD 138. The expression, solubility and activity of AD 127 from the pAD138 construct was very high. Therefore, this chimera was named ‘ClyS’ for Chimeric lysin for Staphylococcus (FIG. 5A).

ClyS (SEQ ID NO:2) contains 280 amino acid residues with a deduced molecular mass of 31956 Da and a theoretical isoelectric point of 9.17, and was purified by two-step column chromatography to >90% homogeneity. ClyS had a molecular mass of approximately 31 kDa by SDS/PAGE (FIG. 6) which was confirmed by gel filtrations chromatography, suggesting that the protein exists as a monomer and is not proteolytically processed (data not shown).

The unit activity of ClyS was defined by measuring the spectrophotometric loss of turbidity, indicative of cell lysis, of S. aureus 8325-4 cells upon adding serial dilutions of ClyS. In our assays, 5 micrograms of ClyS corresponded to 1 U of lytic activity. When 50 U of ClyS was added to exponentially growing 8325-4 cells the OD600 dropped to baseline within 5 min (FIG. 7). To confirm that the observed cell lysis corresponds to cell death, staphylococcal viability was determined by enumerating aliquots from the lytic reaction at various time points. A decrease in viability of approximately 3-logs was observed in 30 min (FIG. 7).

The lytic effect on S. aureus 8325-4 cells exposed to 50 U of ClyS for 1-3 min was visualized by transmission electron microscopy, Typical of lysin activity observed previously, localized degradation of the cell wall was observed at single (FIG. 8A) or multiple sites (FIG. 8B). However, unlike other lysins, the sites of degradation on the cell was not restricted to the septal or polar positions but was randomly distributed. This resulted in extrusions and rupture of the cell membrane (FIG. 8C) and subsequent loss of cytoplasmic contents and formation of cell-ghosts (FIG. 8D).

The effect of pH on the activity of ClyS was determined by measuring the drop in OD₆₀₀ or cell viability at different pH values. We observed that ClyS was active over a wide range of pH values but was most active between pH 9 and 10. However, ClyS retained partial yet significant activity at physiological pH (FIG. 9A). Similarly, the effect of salt concentration on activity of ClyS was also determined, ClyS displayed activity in a wide range of salt concentrations (FIG. 96). While its activity deteriorated above 400 mM NaCl, at physiological concentrations ClyS functioned well.

Muralytic activity of ClyS was tested on a number of bacterial strains representing a variety of species which were divided into sets (Table 1 and FIG. 10). Set I consisted of S. aureus strains including methicillin-sensitive S. aureus (MSSA) and MRSA. ClyS was active against MSSA and MRSA although differences were observed between S. aureus strains. Set II consisted of different species of staphylococci including S. epidermidis, S. simulans and S. sciuri. ClyS was active not only against S. epidermidis including the biofilm-forming strain RP62A but was also active against S. simulans and S. sciuri suggesting that ClyS recognizes an epitope in the cell wall that is present in all staphylococcal cells. Set III consisted of a mix of Gram-positive and Gram-negative bacteria including representatives of group A, B, C and E streptococci, oral streptococcal species including S. gordonli, and S. salivarius, as well as S. uberis, Bacillus cereus, Pseudomonas aeruginosa and E. coli. ClyS exhibited no activity against any of these organisms.

In another embodiment, a chimeric peptide comprises an isolated polypeptide comprising an endopeptidase domain of the S. aureus Twort lysin upstream of the lyphostaphin CWT domain. One example of such a lysin is provided in SEQ ID NO:3 (AD119).

In another embodiment, lytic compositions may comprise a mixture of two or more lysins. The mixture may include a first polypeptide and a second peptide where one or both of the polypeptides may lack a desired level of lytic activity, but the mixture provides desirably specific and effective lytic activity against a bacteria of interest. For example, a composition may include an isolated first polypeptide comprising an endopeptidase domain of the S. aureus Twort lysin upstream of the lyphostaphin CWT domain combined with a second isolated polypeptide comprising an S. epidermidis autolysin amidase domain upstream of the lysostaphin CWT domain. One example of such a composition comprises a mixture of SEQ ID NO:3 (AD119) and SEQ ID NO:4 (AD112).

In some examples, the present disclosure pertains to lytic enzymes as a prophylactic treatment for preventing infection those who have possibly been exposed to S. aureus bacteria, or as a therapeutic treatment for those who have already become ill from the infection. The phage associated lytic enzymes described herein are specific for S. aureus bacteria and preferably effectively and efficiently break down the cell wall of the S. aureus bacteria.

The chimeric lytic enzyme polypeptides described herein may also be employed as a therapeutic agent. The lytic enzyme polypeptides of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the lytic enzyme product hereof is combined in admixture with a pharmaceutically acceptable carrier vehicle. Compositions which may be used for the prophylactic and therapeutic treatment of a S. aureus bacteria infection also includes the shuffled and/or chimeric enzyme and a means of application (such as a carrier system or an oral delivery mode) to the mucosal lining of the oral and nasal cavity, such that the enzyme is put in the carrier system or oral delivery mode to reach the mucosa lining.

In one preferred embodiment, a Staphylococcus chimeric lysin, such as a lysin of SEQ ID NO:2 (ClyS), is administered as an antibacterial composition in combination with a suitable pharmaceutical carrier. In certain embodiments, the amount of the chimeric bacteriophase lysin present is a therapeutically effective amount. “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™, These antimicrobial/pharmaceutical compositions may be administered locally or systemically.

Routes of administration include topical, ocular, nasal, pulmonary, buccal, parenteral (intravenous, subcutaneous, and intramuscular), oral, parenteral, vaginal and rectal. Also administration from implants is possible. The compounds of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and rnicroemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated [see, for example, J Pharm Sci, 88 (10), 955-958 by Finnin and Morgan (October 1999).]

The compounds of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques. The compounds of the invention may also be administered intranasally or orally by inhalation, typically in the form of a aerosol.

Suitable antimicrobial preparation forms are, for example granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, microemulsions, defined as optically isotropic thermodynamically stable systems consisting of water, oil and surfactant, liquid crystalline phases, defined as systems characterized by long-range order but short-range disorder (examples include lamellar, hexagonal and cubic phases, either water- or oil continuous), or their dispersed counterparts, gels, ointments, dispersions, suspensions, creams, aerosols, droplets or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients, diluents, adjuvants or carriers are customarily used as described above. The pharmaceutical composition may also be provided in bandages or in sutures or the like.

Many orthopedic surgeons consider that humans with prosthetic joints should be considered for antibiotic prophylaxis. Late deep infection by S. aureus is a serious complication sometimes leading to loss of the prosthetic joint and is accompanied by significant morbidity and mortality. It may therefore be possible to extend the use of the chimeric bacteriophage lysin described herein (e.g., SEQ ID NO:2) as a replacement for or for use in combination with prophylactic antibiotics in this situation. The chimeric bacteriophage lysin may be administered by injection with a suitable carrier directly to the site of the orthopedic device in situ to clear the infection, or on a surface of the device prior to implantantation. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like.

Prior to, or at the time the enzyme is put in the carrier system or oral delivery mode, it may be desirable for a chimeric peptide described herein to be administered or formulated in a stabilizing buffer environment, maintaining a pH range between about 5.0 and about 7.5. Prior to, or at the time the chimeric peptide is put in the carrier system or oral delivery mode, the enzyme may be in a stabilizing buffer environment for maintaining a suitable pH range, such as between about 5.0 and about 8.0, including a pH of about 5.0, 6.0, 7.0, 8.0 or any pH interval of 0.05 therebetween, or any interval that is a multiple of 0.05 therebetween, including pH values of 5.2, 6.5, 7.4, 7.5 and 8.5.

There are a number of advantages to using lytic enzymes to treat bacterial infections. The modular design of lysins, with their distinct catalytic and binding domains, makes them ideal for domain swapping experiments in which bacterial specificities and catalytic activities can be improved or adapted for use against alternate pathogens. Since the catalytic and binding targets of lysins (peptidoglycan and associated carbohydrates, respectively) are largely essential for viability, lysin resistance will be rare.

“Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc, Preferably, the mammal is human.

The formulations to be used for in vivo administration are preferably sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. Therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes, topical administration, or by sustained release systems. When treating a bacterial exposure or infection, the lytic enzyme may be administered in any suitable fashion, including parenterally or through the oral or nasal cavity.

Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal-experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The use of interspecies scaling in toxicokinetics” In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96.

When in vivo administration of a chimeic peptide lysin is employed, normal dosage amounts may vary from about 10 ng/kg to up to 1000 mg/kg of mammal body weight or more per day, or about 1 μg/kg/day to 10000 mg/kg/day, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is also provided below, as well as in the literature. It is anticipated that different formulations will be effective for different treatment compounds and different disorders, that administration targeting one organ or tissue, for example, may necessitate delivery in a manner different from that to another organ or tissue.

The effective dosage rates or amounts of the chimeric peptide to be administered parenterally, and the duration of treatment will depend in part on the seriousness of the infection, the weight of the patient, the duration of exposure of the recipient to the infectious bacteria, the seriousness of the infection, and a variety of a number of other variables. The composition may be applied anywhere from once to several times a day, and may be applied for a short or long term period. The usage may last for days or weeks. Any dosage form employed should provide for a minimum number of units for a minimum amount of time. The concentration of the active units of a chimeric peptide believed to provide for an effective amount or dosage of enzyme may be in the range of about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 units/nil up to about 10,000,000 units/ml of composition, in a range of about 1000 units/ml to about 10,000,000 units/ml, and from about 10,000 to 10,000,000 units/ml.

Additionally, a number of methods can be used to assist in transporting the enzyme across the cell membrane. The enzyme can be transported in a liposome, with the enzyme be “inserted” in the liposomes by known techniques. Similarly, the enzyme may be in a reverse micelle. The enzyme can also be pegylated, attaching the polyethylene glycol to the non-active part of the enzyme. Alternatively, hydrophobic molecules can be used to transport the enzyme across the cell membrane. Finally, the glycosylation of the enzyme can be used to target specific internalization receptors on the membrane of the cell.

Another preferred embodiment provides for a composition comprising, a Staphylococcus chimeric lysin bacterial binding protein such as a lysin of SEQ ID NO:2 (ClyS), with other lytic enzymes which are useful for sanitizing or decontaminating porous surfaces e.g. textiles, carpeting. Furthermore, the composition of lytic enzymes may be used to decontaminate veterinarian surgical or examination areas, where such areas may be thought to harbor infectious organisms susceptible to the bacteriostatic or bacteriocidal activity.

In a further preferred embodiment, a Staphylococcus chimeric lysin such as a lysin of SEQ ID NO:2 (ClyS) may be combined with other bacteriostatic or bacteriocidal agents useful for decontamination of inanimate solid surfaces suspected of containing infectious bacteria, or for decontamination of porous surfaces.

EXAMPLES Example 1: Identification of Specific Binding Peptides and Development of Chimeric Lysins

We conducted conserved domain searches of Staphylococcus-specific phage and prophage lysin protein sequences in the National Center for Biotechnology Information database. The lysins were classified based on homology to known domains in the database. We identified several lysins including the S. aureus phage phiNM3 lysin (protein accession number YP_908849), S. aureus prophage phi13 amidase (accession number NP_803402), S. aureus prophage MW2 amidase (accession number NP_646703.1), etc. that shared 100% sequence identity with each other and had a conserved CHAP domain within their catalytic domain. However, the C-terminal domain of these lysins did not display homology to any known domains in the database (FIG. 1).

Since the attempts to express a native staphylococcal lysin were unsuccessful, we decided to develop chimeric lysins by taking advantage of the modular nature of lysins. Traditionally, Bacillus-specific lysins are expressed at high levels and are soluble in E. coli. Therefore, our first attempt was to engineer a 16-amino acid peptide (4) that is conserved in several S. aureus-specific lysins (Lu J Z, Fujiwara T, Komatsuzawa H, Sugai M, Sakon J. 2006. Cell wall-targeting domain of glycylglycine endopeptidase distinguishes among peptidoglycan cross-bridges. (Lue et al. (2006) J. Biol. Chem. 281(1):549-58) The catalytic domain of the Bacillus-specific lysin PlyB was used to generate chimera AD 103 (FIG. 4) (SEQ ID NO: 13). The chimeras were tested for expression, solubility and activity. Then the entire C-terminal CWT domain of PlyB (6) was replaced by the putative C-terminal domain of S. aureus phage Twort lysin (8) to obtain AD 105 (SEQ ID NO: 14). This chimera was not active and so we engineered the lysostaphin CWT domain (10) downstream of PlyB-catalytic domain (2) to get AD 107 (SEQ ID NO:15). Although this chimera had expression, the solubility was poor and there was no activity. The next step was to engineer a S. epidermidis autolysin amidase domain (12) upstream of the lysostaphin CWT domain (10) which resulted in AD 112 (SEQ ID NO:4). AD 112 expressed very well and the protein was also very soluble but there was no lytic activity. However, we observed that the S. aureus cells clumped when exposed to AD 112. Since the lysostaphin catalytic domain (an amidase) (10) in AD 112 was of bacterial origin, we attempted to engineer a phage-derived catalytic domain upstream of the lysostaphin CWT. For this, the endopeptidase domain in Twort lysin (14) was used to construct chimera AD 119 (SEQ ID NO:3). We observed poor expression for AD 119 but the chimera was soluble. Although in our lytic assays AD 119 alone did not show significant activity, when combined with chimera AD 112 the activity was significantly enhanced. Since we identified phiNM3 CWT domain from our conserved domains searches and observed that phiNM3 CWT exhibited Staphylococcus-specific binding, we constructed chimeras AD 126 (SEQ ID NO:16) and AD 127 (SEQ ID NO:2) by engineering S. epidermidis autolysin amidase (12) and Twort lysin endopeptidase (14) domains upstream of phiNM3 CWT domain (2) (SEQ ID NO:1), respectively. Chimera AD 126 had no expression or activity but AD 127 was soluble and had very high activity but low expression (FIG. 4). To overcome low expression of AD 127 construct, the entire chimera gene was cloned into expression vector pJML6 to generate pAD 138. The expression, solubility and activity of AD 127 from the pAD138 construct was very high. Therefore, this chimera was named ‘ClyS’ for Chimeric lysin for Staphylococcus. The amino acid sequence for ClyS (i.e., SEQ ID NO:2) is provided in FIG. 5A.

Example 2: Construction of the ClyS Chimeric Lysin

Bacterial strains (Table 1) were stored at −80° C. routinely grown at 37° C. Staphylococcal strains used in this study were grown in Trypticase Soy Broth (TSB) media, streptococcal strains were grown in THY (Todd-Hewitt broth, 1% wt/vol yeast extract) media, B. cereus and P. aeruginosa were grown in BHI (Brain Heart Infusion) media while E. coli was cultivated in LB (Luria Bertani) media.

The chimeric lysin was constructed by amplifying and ligating individual domains from respective genes. For this, the Twort endopeptidase domain was PCR amplified from plasmid pCR2.1 plyTW which contains the entire lysin (plyTW) gene using primers TW-Endo-Ncol-F: 5′-CTAGCCATGGAAACCCTGAAACAAGCAG-3′ (SEQ ID NO:5) and TW-Endo-Pstl-R: 5′-ACATGCTGCAGAACCATATTGTAATTAATATTAGTTCTATC-3′(SEQ ID NO:6). The cell wall targeting (CWT) domain was PCR amplified from S. aureus strain 8325 genomic DNA, using primers NM3-CBD-Pstl-F: 5′-ACATGCTGCAGGGTAAATCTGCAAGTAAAATAACAG-3′ (SEQ ID NO:7) and NM3-CBD-Hind-R: 5′-CCCAAGCTTAAAACACTTCTTTCACAATCMTCTC-3′(SEQ ID NO:8). The two PCR amplicons were ligated using the Pstl restriction endonuclease site. The ligated product was cloned into pBAD24 vector using the Ncol-HindIII cloning sites to generate recombinant plasmid pAD127. In the second step, the entire DNA fragment corresponding to clyS was PCR amplified from pAD124 using primers NM3-Lys-Xba-F: 5′-CTAGTCTAGAGGTGGAATAATGAAAACATACAGTGAAGCAAG-3′ (SEQ ID NO:9) and primer NM3-CBD-Hind-R(SEQ ID NO:8). The PCR product was cloned into expression vector pJML6 to generate pAD138. The sequence of ClyS was confirmed by sequencing. The recombinant plasmid pAD138 was transformed into E. coli DH5a cells.

Example 3: Overexpression and Purification of ClyS

ClyS was induced overnight from E. coli DH5a (pAD138) cells with lactose (10 g/500 ml final concentration) at 30° C. Cells were harvested by centrifugation, resuspended in buffer A (20 mM phosphate buffer (PB), 1 mM DTT (dithiothreitol)) and lysed by an EmulsiFlex-05 high pressure homogenizer (Avestin) at 400 C. The lysates were cleared by centrifugation (2×50,000×g) for 30 minutes at 4° C. and the supernatant applied to a CM-sepharose column (Amersham Pharmacia, Piscataway, N.J.). ClyS was eluted with buffer A+1M NaCl using a linear gradient of 0-50% B in 15 columns volumes. Fractions were analyzed for lytic activity as previously described (Daniel et al, 2001). Fractions displaying lytic activity were pooled and dialyzed overnight against buffer B (PB, 1 mM DTT, 50 mM NaCl). The dialyzed sample was applied to a hydroxylapatite (MacroPrep TypeII 40 μm, BioRad) column and eluted with elution buffer (500 mM PB+50 mM NaCl+1 mM DTT) using a linear gradient of 0-100% B in 20 columns volumes. The fractions were analyzed by SDS-PAGE and for lytic activity. Active clean fractions of ClyS were pooled and dialyzed against buffer B. Protein concentration was determined with the BCA method (Sigma, St. Louis, Mo.).

Example 4: Quantification of ClyS Activity

ClyS activity was measured as previously described (Daniel et al, 2001), with some modifications. Briefly, S. aureus strain 8325-4 was grown to an OD₆₀₀ of 0.25-0.3, centrifuged, and resuspended in PB to a final OD₆₀₀ of 0.8-1.0. Two-fold serial dilutions of purified ClyS (1000) were added to 100 μl of bacterial suspension in 96-well plates (Costar) and the decrease in OD600 was monitored by a Spectramax Plus 384 spectrophotometer (Molecular Devices) over 30 min at 37° C. ClyS activity in units per milliliter was defined as the reciprocal of the highest dilution of lysin that decreased the absorbance by 50% in 15 minutes.

Example 5: Measuring In Vitro ClyS Activity

The viability assay of ClyS was tested as previously described (Nelson et al, 2001). Briefly, log phase cultures of S. aureus strain 8325-4 were resuspended in PB to OD₆₀₀ of 0.8-1.0. 50 U of ClyS or the corresponding volume of PB was added to bacterial cells and aliquots were removed, serially diluted, and plated at 1, 5, 10, 30, and 60 minutes to assess the viability of the treated and control cells. All experiments were performed in triplicate. The activity of ClyS on various bacterial strains was tested as described previously (Schuch et al, 2002). Briefly, log phase bacterial cells were treated with 50 U of ClyS at 37° C. for 15 minutes. The samples were serially diluted and plated. Control experiments with the addition of phosphate buffer (pH 7.0) were performed under the same conditions.

Example 6: Measuring ClyS Activity as a Function of pH and Salt Profile

The effect of pH on ClyS activity was determined as previously described using the universal buffer system pH 4-10 (Yoong et al). Briefly, log phase 8325-4 cells were resuspended in the universal buffer system and incubated with 50 U of ClyS for 15 minutes. The final pH of each reaction was checked by pH paper. The samples were serially diluted and plated. In controls, PB replaced ClyS.

Similarly the effect of salt concentration on the lytic activity of ClyS was determined by incubating 50 U of ClyS with log phase 8325-4 cells in PB containing Nadi at a final concentration of 25-500 mM for 15 minutes. The samples were serially diluted and plated to determine the viability counts.

Example 7: Microscopy of ClyS

S. aureus strain 8325-4 was grown to log-phase, centrifuged and resuspended in PBS to an absorbance at 600 nm of 1.0. The bacterial suspension was incubated with 50 U of ClyS at room temperature. The lytic reaction was terminated after 1 minute and 5 minutes by adding glutaraldehyde (final concentration 2.5%). The suspension was pelleted by centrifugation and overlaid with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The samples were then postfixed in 1% osmium tetroxide, block stained with uranyl acetate and processed according to standard procedures by The Rockefeller University Electron Microscopy Service.

Flourescent labeling and binding analyses were performed on phiNM3 CWT. S. aureus strain 8325-4 genomic DNA was used to amplify the putative CWT of phiNM3 lysin using primers NM3-FWD CATGCCATGGGTAAATCTGCAAGTAAAATAACAG-3′ (SEQ ID NO:10) and NM3-REV 5′-CCCAAGCTTAAAACACTTCTTTCACAATCAATCTC-3′(SEQ ID NO:11). The resulting amplicon was cloned into the arabinose-inducible expression vector pBAD24. Positive clones containing the insert were confirmed by sequencing. The approximately 10-kDa phiNM3 CWT protein was expressed and the protein was purified in one step by cation-exchange chromatography. The purified protein (1 mg/ml) was incubated with 10 μl of FITC (1 mg/ml) for 1 hour. Excess FITC was removed on a desalting column. The labeled-protein (50 μg) was incubated with bacterial cells for 10 minutes, washed 3× with phosphate-buffered saline (pH 7.4) and observed under fluorescence microscope.

Example 8: Measuring In Vivo Activity of ClyS

MRSA strain would be grown to log-phase, centrifuged and resuspended to a predefined titer of about 1010 cfu/ml. For intranasal infection, 6-wk-old female C57BL/6J, outbread Swiss or BALB/c mice (weight range 22 to 24 g, Charles River Laboratories, Wilmington, Mass.) would be anesthetized with a mixture of ketamine (Fort Dodge Animal Health, Fort Dodge, Iowa, 1.2 mg/animal) and xylazine (Miles Inc., Shawnee Mission, Kans., 0.25 mg/animal), and inoculated with 15 μl of the bacterial suspension per nostril (n=10). The animals would be divided into 2 groups and administered various concentrations of ClyS or sterile saline intraperitoneally six hours after infection and every six hours thereafter for 3 days. The survival rate for each group would be observed up to 7 days post infection. For intraperitoneal infection, mice would be infected intraperitoneally with 100 μl of the bacterial suspension (n=10), The animals would be divided into 2 groups and administered various concentrations of ClyS or sterile saline intraperitoneally six hours after infection and every six hours thereafter for 3 days. The survival rate for each group would be observed up to 7 days post infection.

Example 9. The Linker Region by Itself does not Confer Solubility to a Chimera

Since the ClyS construct was the only chimera that was highly soluble and active against staphylococci, we hypothesized that the linker region comprising of amino acid residues 142 through 185 of ClyS may be crucial for solubility. We had previously cloned and expressed the native phiNM3 lysin and observed that the protein was insoluble. To test this hypothesis, we replaced the endopeptidase domain of ClyS with the amidase domain of phiNM3 lysin upstream of the linker region of ClyS (ami-link-ClyS) and expressed the chimera (data not shown). We observed that similar to the native phiNM3 lysin, the ami-link-ClyS chimera was insoluble and expressed as inclusion bodies. We also tested the lysates of ami-link-ClyS for activity against staphylococci and did not observe any lytic activity confirming that the protein was insoluble and therefore inactive. Thus, it is the unique combination of the N and C terminal domains that are the subject of this patent that allow for a soluble complex to occur and behave as described herein.

Example 10. In Vivo Nasal Decolonization of MRSA by ClyS

Carriage of both MSSA and MRSA in the human anterior nares is the major reservoir for S. aureus infection. Studies have shown that roughly 80% of the population could be nasally colonized by S. aureus, and that colonization can be an increased risk factor for developing other more serious S. aureus infections (Kluytmans, J., A. van Belkum. 1997. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev 10(3): 505-20). Elimination of nasal carriage in the community or in the hospital setting thus could possibly reduce the risk of infection and slow the spread of drug resistant S. aureus (Kluytmans et al. (1997)). To study the potential of ClyS to reduce MRSA colonization of the nasal mucosa, C57BL/6J mice were intranasally inoculated with ˜2×10⁷ of a spontaneously streptomycin resistant strain of MRSA (191-SM^(R)). Twenty-four hours post-infection mice were administered three doses hourly of either phosphate buffered saline (control) or ClyS (960 μg) into the nasal passages. One hour after the last treatment, mice were sacrificed and bacteria colonies were enumerated on Spectra MRSA agar, (a selective chromogenic medium developed to diagnostically detect MRSA nasal colonization) and Columbia blood agar. No significant differences in CFU were obtained between plating to Spectra MRSA agar or Columbia blood agar (Data not shown) Three independent experiments were performed to evaluate a total 20 mice for each treatment group (FIG. 11). Compared to the buffer alone control (Avg. 12,273 CFU/cavity), ClyS treatment (Avg. 1198 CFU/cavity) significantly (p<0.001) reduced the mean CFU on the nasal mucosa.

Example 11. ClyS Treatment of Systemic MRSA Infections

In order to assess whether ClyS treatment could prevent death resulting from systemic MRSA infections, 4 week old FVB/NJ mice were intraperitonally injected with ˜5×10⁵ CFU of the community-acquired MRSA strain MW2 in 5% mucin. Preliminary experiments determined that 5×10⁵ CFU was 10× the LD₁₀₀ dose for a twenty-four hour period. Furthermore, within 3 hours of IP injection the MRSA infection was systemic. i.e., MRSA were recovered in high numbers from heart, liver, spleen, and kidney (data not shown). Treatment occurred three hours post-infection, with either 20 mM phosphate buffer or 1 mg of ClyS in 20 mM phosphate buffer injected IP (intraperitoneally). Mice were then monitored for survival over ten days. The results from three independent experiments were combined (ClyS treatment, n=16; buffer treatment, n=14) and the mice survival data plotted with a Kaplan Meier Survival curve (FIG. 12), Within twenty-four hours all of the control mice died of bacterial sepsis, while only 2/16 of ClyS treated mice died at forty-eight hours, and the remaining mice (14/16, 88%) survived over the course of the experiments (FIG. 12).

Example 12. ClyS Showed Synergistic Interaction with Vancomycin and Oxacillin

We used the checkerboard broth-microdilution assay to test the interaction of ClyS with vancomycin and with oxacillin. The vancomycin MIC for VISA strain Mu50 was 8 μg/ml and the oxacillin MIC for MRSA strain COL was 32 μg/ml, while the ClyS MIC was 6 and 8 U/ml for both strains tested (Mu50 and COL respectively). Isobolograms for ClyS with vancomycin and ClyS with oxacillin was plotted by transcribing the enzyme concentrations along the inhibitory line on the microtiter plate in an x/y plot. The shape of the curves for both interactions were characteristic of a synergistic interaction (FIG. 13) and were further confirmed by calculating the ΣFICI for both interactions which was s0.5.

Example 13. In Vivo Synergy of Oxacillin and ClyS in the Treatment of Systemic MRSA Infections

In vitro experiments showed that ClyS acted synergistically with oxacillin (FIG. 14). To determine if this effect could be seen in our systemic MRSA infection model, FVB/NJ mice were intraperitonally injected with ˜5×10⁵ CFUs of the MRSA strain MW2 as above. Three hours post infection mice were treated in parallel, with a lower IP dose of 130 μg/mouse of ClyS combined with different concentrations of oxacillin (10-100 μg/mouse) or buffer alone controls. Preliminary experiments determined that an ED₃₀ dose of ClyS (130 μg/mouse) had minimal efficacy to evaluate the effect of combinatorial treatment with oxacillin (data not shown). Mice were monitored for survival for 10 days and the results of 5 independent experiments were combined and plotted in a Kaplan Meier Survival curve (FIG. 14). While only 30% (6/20 alive) to 35% (8/23 alive) of mice survived with individual treatments of either 130 μg/mouse of ClyS or 100 μg/mouse of oxacillin, respectively, neither differed significantly from the survival rate of the buffer alone control, 13% (2/15 alive). Conversely, a single dose of the combined treatment of intraperitoneal injected ClyS (130 μg) with either 0 μg or 50 μg of intramuscular injected oxacillin significantly increased mouse survival (80%, 8/10 alive; 82%, 18/22 alive respectively) compared to the individual treatments and buffer alone (FIG. 14).

Example 16. Modification of ClyS

The G¹⁰⁰ residue of ClyS (SEQ ID NO:2) was changed to a proline by site directed mutagenesis (creating pClyS). When the purified pClyS molecule (SEQ ID NO: 17) was subjected to stability studies at 21° C. for 5 days, the pClyS was found to be significantly more stable in the presence of 5 mM DTT the native ClyS with or without OTT (FIG. 15).

While the invention has been described and illustrated herein by reference to various specific materials, procedures, and examples, it is understood that the invention is not restricted to the particular materials, combinations of materials, and procedures selected for that purpose. Numerous variations of such details can be implied and will be appreciated by those skilled in the art. 

1.-23. (canceled)
 24. A chimeric bacteriophage lysin comprising a catalytic domain of a first Staphylococcus phage lysin bound to the binding domain of another second Staphylococcus phage lysin, wherein the binding domain has the polypeptide sequence of SEQ ID NO:1 or a variant of SEQ ID NO:1 having at least 90% amino acid sequence identity to SEQ ID NO:1 and capable of binding specifically to staphylococci, wherein the chimeric lysin comprises the polypeptide sequence of SEQ ID NO: 2 or a variant of SEQ ID NO: 2 having at least 90% amino acid sequence identity to SEQ ID NO: 2, and wherein said chimeric lysin or variant has killing activity against Staphylococcus aureus and is capable of binding specifically to staphylococci.
 25. The chimeric lysin of claim 24, wherein the Gly 166 residue of SEQ ID NO: 2 is changed to a Proline and wherein the variant has at least 90% amino acid identity to SEQ ID NO:
 2. 26. The chimeric lysin of claim 25, where a variant of SEQ ID NO:2 comprising a Proline at residue 166 of SEQ ID NO:2 is more stable than the native SEQ ID NO:2 in the presence of DTT.
 27. The chimeric lysin of claim 24, wherein the variant of SEQ ID NO:2 has at least 95% amino acid sequence identity to SEQ ID NO: 2 and is capable of binding specifically to staphylococci and has killing activity against Staphylococcus aureus.
 28. The chimeric lysin of claim 25, wherein the variant of SEQ ID NO:2 has at least 95% amino acid sequence identity to SEQ ID NO: 2 and is capable of binding specifically to staphylococci and has killing activity against Staphylococcus aureus.
 29. The chimeric lysin of claim 25, wherein the lysin has a SEQ ID NO:2 variant polypeptide sequence corresponding to SEQ ID NO: 2 wherein the Gly 166 residue is changed to a Proline.
 30. The chimeric lysin of claim 25, where the binding domain comprises the polypeptide sequence of SEQ ID NO:1.
 31. The chimeric lysin of claim 25, where the chimeric lysin has killing activity against methicillin-resistant and methicillin-sensitive strains of S. aureus.
 32. The chimeric lysin of claim 25, having killing activity against multiple staphylococcal species.
 33. The chimeric lysin of claim 25, where the lysin kills Staphylococcus epidermidis.
 34. The chimeric lysin of claim 25, having killing activity against Staphylococcal bacteria over a pH range of about 4-10.
 35. An anti-microbial composition for sanitizing or decontaminating porous or non-porous surfaces comprising the chimeric lysin of claim
 25. 36. A method for decontaminating inanimate surfaces suspected of containing infectious staphylococci bacteria comprising treatment of said surfaces with a bacteriocidal or bacteriostatically effective amount of the composition of claim
 35. 37. A pharmaceutical composition comprising the chimeric lysin of claim 25 and a pharmaceutically acceptable carrier.
 38. The pharmaceutical composition of claim 37 further comprising one or more bacteriostatic or bacteriocidal agents.
 39. The pharmaceutical composition of claim 37 further comprising one or more antibiotic.
 40. The pharmaceutical composition of claim 37 which is formulated for topical, ocular, nasal, pulmonary, buccal, parenteral, oral, vaginal or rectal administration.
 41. A method of treating a mammal with a staphylococcal infection comprising administering to said mammal the chimeric bacteriophage lysin of claim
 25. 42. A method of treating a mammal with a staphylococcal infection comprising administering to said mammal the pharmaceutical composition of claim
 37. 43. A method of treating a mammal with a staphylococcal infection comprising administering to said mammal the pharmaceutical composition of claim
 38. 